Structural insights into conformational changes of a cyclic nucleotide-binding domain in solution from Mesorhizobium loti K1 channel

Abstract

Cyclic nucleotide-sensitive ion channels, known as HCN and CNG channels, are activated by binding of ligands to a domain (CNBD) located on the cytoplasmic side of the channel. The underlying mechanisms are not well understood. To elucidate the gating mechanism, structures of both the ligand-free and -bound CNBD are required. Several crystal structures of the CNBD from HCN2 and a bacterial CNG channel (MloK1) have been solved. However, for HCN2, the cAMP-free and -bound state did not reveal substantial structural rearrangements. For MloK1, structural information for the cAMP-free state has only been gained from mutant CNBDs. Moreover, in the crystal, the CNBD molecules form an interface between dimers, proposed to be important for allosteric channel gating. Here, we have determined the solution structure by NMR spectroscopy of the cAMP-free wild-type CNBD of MloK1. A comparison of the solution structure of cAMP-free and -bound states reveals large conformational rearrangement on ligand binding. The two structures provide insights on a unique set of conformational events that accompany gating within the ligand-binding site.NMR solution structure | apo state | ligand removal method | potassium channel I on channels activated by cyclic nucleotides play key roles in neuronal excitability and sensory signaling. They belong to two subfamilies: cyclic nucleotide-gated (CNG) channels, and hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels (1-4). CNG channels are voltage independent and require cyclic nucleotides to open, whereas HCN channels are activated by hyperpolarization, and their activity is modulated by cyclic nucleotides. In both channels, ligand binding to an intracellular cyclic nucleotide-binding domain (CNBD) promotes the opening of the channel by conformational changes in the CNBD that propagate via a connecting linker (C linker) to transmembrane segment S6. However, the activation mechanisms are poorly understood in part because the nature of the pore gate is not known precisely (4).Understanding of the structural rearrangements underlying gating has been greatly advanced by several crystal structures of isolated CNBDs from HCN channels (5-8) and a prokaryotic K þ -selective CNG channel, designated MloK1 (9-11). The MloK1 channel consists of four identical subunits, each encompassing six transmembrane domains (S1-S6), a "GYG" signature sequence for K þ selectivity, and a conserved CNBD. However, the C linker (approximately 20 residues) is much shorter than the C linker of mammalian CNG and HCN channels (approximately 80 residues) that is important for relaying the binding signal from the CNBD to the channel gate (12-17). In the crystal, the CNBDs from HCN channels are arranged in tetramers, where neighboring C linkers contribute most contacts between subunits (6, 8), whereas the MloK1 CNBD forms a dimer; the dimer interface between the short C linker has been proposed to be involved in channel gating (9, 11). The solution structure solved by nuclear magnetic resonance (NMR) spectroscopy...

Double electron–electron resonance reveals cAMP-induced conformational change in HCN channels

“…In particular, the NMR structures of the CNBD of the prokaryotic K + channel MloK1 in the absence and presence of cAMP demonstrate a similar movement of the B-and C-helices subsequent to agonist binding (39,40). However, the movement of the C-helix is somewhat larger than reported here.…”

Structural snapshot of cyclic nucleotide binding domains from cyclic nucleotide-sensitive ion channels

“…Helix αC is disordered or partially unfolded and in some structures no electron density could be observed for this particular region (Clayton et al, 2004;Altieri et al, 2008). However, the solution structure of the wild-type MloK1 CNBD in the apo-state (Figure 2) shows that the position of helix αC is well defined and adopts a single orientation (Schünke et al, 2011). Moreover, comparison of the wild-type cAMP-free and -bound CNBD solution structures shows large conformational rearrangements of the complete helical bundle on ligand-binding.…”

“…Addressing the question whether the CNBD of eukaryotic HCN or CNG channels undergo structural rearrangements upon ligand-binding in a manner that differs to the bacterial MloK1 CNBD (Clayton et al, 2004;Altieri et al, 2008;Schünke et al, 2009;Mari et al, 2011;Schünke et al, 2011) is not possible, because only structural information of one ligand-free eukaryotic CNBD is available (Taraska et al, 2009) in contrast to multiple structures in the ligandbound state (see Table S1). …”

A KcsA/MloK1 Chimeric Ion Channel Has Lipid-dependent Ligand-binding Energetics

“…MloK1 contains the signature GYG selectivity filter sequence found in HCN channels, but the "voltage sensor domain" lacks the arginine repeats that allow voltage sensing (26). Crystal structures of the transmembrane and CNBD domains of MloK1 have been determined separately (24,(27)(28)(29). The transmembrane domain has a structure similar to that of other voltage-gated potassium channels, but the absence of the CNBDs precludes insight into ligand modulation of MloK1.…”

“…The transmembrane domain has a structure similar to that of other voltage-gated potassium channels, but the absence of the CNBDs precludes insight into ligand modulation of MloK1. The structures of the isolated MloK1 CNBDs, determined with and without ligand, show that cAMP binding leads to a clamping down of the C-terminal helix over the ligandbinding pocket and repositioning of the N-terminal helix, which in the full-length channel continues with the inner helix of the transmembrane domain (28). In addition, a 16-Å electron microscopy-derived structure of full-length MloK1 in the presence of ligand reveals a symmetrical arrangement of the CNBDs around the transmembrane domain (30).…”